Prefrontal Cortex Astroglia Modulate Anhedonia-like Behavior

Abstract

Reductions of astroglia expressing glial fibrillary acidic protein (GFAP) are consistently found in the prefrontal cortex (PFC) of patients with depression and in rodent chronic stress models. Here, we examine the consequences of PFC GFAP+ cell depletion and cell activity enhancement on depressive-like behaviors in rodents. Using viral expression of diphtheria toxin receptor in PFC GFAP+ cells, which allows experimental depletion of these cells following diphtheria toxin administration, we demonstrated that PFC GFAP+ cell depletion induced anhedonia-like behavior within 2 days and lasting up to 8 days, but no anxiety-like deficits. Conversely, activating PFC GFAP+ cell activity for 3 weeks using designer receptor exclusively activated by designer drugs (DREADDs) reversed chronic restraint stress-induced anhedonia-like deficits, but not anxiety-like deficits. Our results highlight a critical role of cortical astroglia in the development of anhedonia and further support the idea of targeting astroglia for the treatment of depression.

Fig.1:. Cortical Glial Fibrillary Acidic Protein (GFAP)+ cell ablation induces behavioral deficits.

(a) Schematic representation of the AAV5-GFP-DIOCMV-DTRflag viral construct designed to conditionally induce expression of diphtheria toxin (DT) receptor (DTR) in GFAPcre+ cells. (b) Experimental timeline illustrating the sequence of behavioral testing on GFAPcre+ mice infused in the PFC with the AAV5-GFP-DIOCMV-DTRflag: sucrose test (ST), elevated plus maze (EPM), open field (OF), novelty suppressed feeding (NSF), locomotor activity (LM), novelty induced hypophagia (NIH), and forced-swim test (FST). Following DT administration (circle - 0, square - 0.1, triangle - 5, and inverted triangle - 20μg/kg), percent sucrose consumption in the ST was measured on day 1 (c), day 2 (d), day 3 (e) for 24 hours, and on day 4 (f) for 1 hour after 16hr fluid deprivation. Mice were also tested in the EPM (g), OF (h), NSF (i), and NIH (j). On day 8, mice percent sucrose consumption in the ST was measure (k) and were tested in the FST (l). Additional behavioral assessments (LM, water intake, and homecage latency to feed and drink) can be found in Supplementary Table 2. Overall z-score of anxiety-like z-score (m) and anhedonia-like z-score (n) were calculated for each animal and group. Data are presented as individual animals and mean ± SEM. *p< .05, **p< .01, and ***p<.001 as compared to DT0 group.

Fig.2 –. Cell specificity of diphtheria toxin receptor (DTR) expression and cortical glial fibrillary acidic protein (GFAP)+ cell ablation.

In GFAPcre− mice infused with AAV5-GFP-DIOCMV-DTRflag, the CMV promoter drives the expression of GFP as shown in a representative 2x image of GFP staining at the infection site (a). (b) High magnification of (a)-insert, with white arrows indicating GFP expressing cells which also express GFAP (c). In GFAPcre+ mice infused with AAV5-GFP-DIOCMV-DTRflag, the CMV promoter drives the expression of DTRflag. GFP and Flag immunostaining showed that some cells express very low levels of GFP (white arrow) (d), most infected cells express only DTRflag (empty arrow) (e), and cells expressing GFP also express flag (white arrow) (f). (b-f) Scale = 50um. (g) DTRflag cell density per mm2 was quantified in GFAPcre+ mice infused with AAV5-GFP-DIOCMV-DTRflag and treated or not with DT (circle - 0, square - 0.1, triangle - 5, and inverted triangle - 20μg/kg). Data are presented as individual animals and mean ± SEM. *p< .05 as compared to DT0 group.

Fig.3:. Virus cell specificity and enhancement of activity in PFC GFAP+ cells infused with AAV5-GFAP-hM3D(Gq)-mCherry virus.

(a) Immunohistochemistry confirming AAV5-Zac2.1 gfaABC1D-lck-GCaMP6f virus specificity in PFC GFAP+ cells. White arrows indicate infected cells express GCaMP (green) and are co-labeled with GFAP+ cells (red). Yellow arrows indicate that NeuN+ cells (blue) are not co-labeled with either GFAP or GCaMP. Scale = 20um (b) Immunohistochemistry confirming AAV5-GFAP-hM3D(Gq)-mCherry virus specificity in PFC GFAP+ cells. White arrows indicate infected cells express mCherry (red) and are co-labeled with GFAP+ cells (green). Yellow arrows indicate that NeuN+ cells (blue) are not co-labeled with either GFAP or mCherry. Scale = 20um (c) Representative 30 second trace recording of calcium transients before and after acute CNO administration. Dotted black line: Z-score = 0. Orange dots indicates local maxima peaks that are 3 median absolute deviations (MAD) above the median. Y axis scale represents 10% z-score deltaF/F and X axis scale represents 5 seconds. (d) Peak frequency of calcium transients recorded for 30mins before and after acute administration of saline or clozapine-n-oxide (CNO) in animal infused with both viruses. Data represents peak frequency for individual recording for each animal before and after injection. *p< .05 compared to before injection conditions.

Fig.4:. Cortical Glial Fibrillary Acidic Protein (GFAP)+ cell activity enhancement reverses chronic restraint stress (CRS)-induced anhedonia- but not anxiety-like behavior.

(a) Experimental timeline illustrating the sequence of behavioral testing on mice infused in the PFC with the AAV5-GFAP-hM3D(Gq)-mCherry virus and subjected to CRS or not and treated with clozapine-n-oxide (CNO) or not: sucrose test (ST), PhenoTyper test (PT), coat state (CS), novelty suppressed feeding (NSF), novelty induced hypophagia (NIH), and locomotor activity (LM). (b-e) Behavioral assessment of mice subjected to 2 weeks of CRS or no CRS on sucrose consumption (b), coat state (c), residual avoidance (RA) (d), and hourly time spent in the shelter zone recorded in the PT (e). (f-k) Behavioral assessment of mice subjected to 5 weeks of CRS or no CRS and treated or not with CNO for the last 3 weeks of CRS on sucrose consumption (f), coat state (g), residual avoidance (RA) (h), and hourly time spent in the shelter zone recorded in the PT (i), NSF (j), and NIH (k). Overall z-score of anxiety-like z-score (l) and anhedonia-like z-score (m) were calculated for each animal and group. (n) Fluorescence intensity of Fosb per cell was quantified in 10 GFAP+/mCherry+ cells per hemisphere per animal. Females are shown as triangles and males as circles. Data are presented as mean ± SEM. Between group significant differences are marked as *p< .05, **p< .01, ***p<.001, and ****p<.0001: No CRS+No CNO vs. CRS+No CNO; #p< .05, ##p< .01, ###p<.001,and ####p<.0001: No CRS+CNO vs. CRS+CNO; &p< .05, &p< .01, &p<.001, and &p<.0001: CRS+No CNO vs. CRS+CNO; %p< .05, %p< .01, %p<.001, and %p<.0001: No CRS+No CNO vs. No CRS+CNO.